Express Efflux Pumps Research Articles

Enhancing PDT efficacy in NMIBC: Efflux inhibitor mediated improvement of PpIX levels and efficacy of the combination of PpIX-PDT and SO-cleavable prodrugs.

Protoporphyrin IX (PpIX)-based photodynamic therapy (PDT) has shown limited efficacy in nonmuscle-invasive bladder cancer (NMIBC). To improve PDT efficacy, we developed singlet oxygen-cleavable prodrugs. These prodrugs, when combined with PpIX-PDT, induce cancer cell death through both PDT and drug release mechanisms. Inhibition of PpIX efflux was reported to be an effective strategy to improve PpIX-PDT in certain cancer cells. Our main goal was to investigate whether adding an efflux inhibitor to the combination of PpIX and prodrugs can improve the PpIX levels in bladder cancer cells and the release of active drugs, thus improving the overall efficacy of the treatment. We treated bladder cancer cell lines with lapatinib and evaluated intracellular PpIX fluorescence, finding significantly increased accumulation. Combining lapatinib with prodrugs led to significantly reduced cell viability compared to prodrugs or PpIX-PDT alone. The effect of lapatinib depended on the expression level of the efflux pump in bladder cancer cells. Interestingly, lapatinib increased paclitaxel (PTX) prodrug uptake by threefold compared to prodrug alone. Adding an efflux inhibitor (e.g., lapatinib) into bladder instillation solutions could be a straightforward and effective strategy for NMIBC treatment, particularly in tumors expressing efflux pumps, with the potential for clinical translation.

Promoter regulatory mode evolution enhances the high multidrug resistance of tmexCD1-toprJ1.

Antibiotic resistance could rapidly emerge from acquiring the mobile antibiotic resistance genes, which are commonly evolved from an intrinsic gene. The emergence of the plasmid-borne mobilized efflux pump gene cluster tmexCD1-toprJ1 renders the last-resort antibiotic tigecycline ineffective, although its evolutionary mechanism remains unclear. In this study, we investigate the regulatory mechanisms of the progenitor NfxB-MexCD-OprJ, a chromosomally encoded operon that does not mediate antibiotic resistance in the wild-type version, and its homologs, TNfxB1-TMexCD1-TOprJ1 mediating high-level tigecycline resistance, and TNfxB3-TMexCD3-TOprJ1. Mechanistic studies demonstrated that in nfxB-mexCD-oprJ, MexCD expression was under a weaker promoter, PmexC and inhibited by a strong repressor NfxB. For tmexCD1-toprJ1, TMexCD1 was highly expressed owing to the presence of a strong promoter, PtmexC1, and an inactive suppressor, TNfxB1, with a T39R mutation that rendered it unable to bind to promoter DNA. In tnfxB3-tmexCD3-toprJ1b, TMexCD3 expression was intermediate because of the local regulator TNfxB3, which binds to two inverted repeat sequences of PtmexC. Additionally, TNfxB3 exhibited lower protein expression and weaker DNA binding affinity than its ancestor NfxB, together with their promoter activities difference explaining the different expression levels of tmexCD-toprJ homologs. Distinct fitness burdens on these homologs-carrying bacteria were observed due to the corresponding expression level, which might be associated with their global prevalence. In summary, our data depict the mechanisms underlying the evolution and dissemination of an important mobile antibiotic resistance gene from an intrinsic chromosomal gene.IMPORTANCEAs antibiotic resistance seriously challenges global health, tigecycline is one of the few effective drugs in the pipeline against infections caused by multidrug-resistant pathogens. Our previous work identified a novel tigecycline resistance efflux pump gene cluster tmexCD1-toprJ1 in animals and humans, together with its various variants, a rising clinical concern. Herein, this study focused on how the local regulation modes of tmexCD1-toprJ1 evolved to a highly expressed efflux pump. Through comparative analysis between three tnfxB-tmexCD-toprJ homologs and their progenitor nfxB-mexCD-oprJ, modes, we demonstrated the evolutionary dynamics from a chromosomal silent gene to an active state. We found the de-repression of the local regulator and an increase of the promoter activity work together to promote a high production of drug efflux machines and enhance multidrug resistance. Our findings revealed that TMexCD1-TOprJ1 adopts a distinct evolutionary path to achieve higher multidrug resistance, urgently needing tight surveillance.

Modulatory role of SmeQ in SmeYZ efflux pump-involved functions in Stenotrophomonas maltophilia.

SmeYZ is a constitutively expressed efflux pump in Stenotrophomonas maltophilia. Previous studies demonstrated that: (i) smeYZ inactivation causes compromised swimming, oxidative stress tolerance and aminoglycoside resistance; and (ii) the ΔsmeYZ-mediated pleiotropic defects, except aminoglycoside susceptibility, result from up-regulation of entSCEBB'FA and sbiAB operons, and decreased intracellular iron level. To elucidate the modulatory role of SmeQ, a novel cytoplasmic protein, in ΔsmeYZ-mediated pleiotropic defects. The presence of operons was verified using RT-PCR. The role of SmeQ in ΔsmeYZ-mediated pleiotropic defects was assessed using in-frame deletion mutants and functional assays. A bacterial adenylate cyclase two-hybrid assay was used to investigate the protein-protein interactions. Gene expression was quantified using quantitative RT-PCR (RT-qPCR). SmeYZ and the downstream smeQ formed an operon. SmeQ inactivation in the WT KJ decreased aminoglycoside resistance but did not affect swimming and tolerance to oxidative stress or iron depletion. However, smeQ inactivation in the smeYZ mutant rescued the ΔsmeYZ-mediated pleiotropic defects, except for aminoglycoside susceptibility. In the WT KJ, SmeQ positively modulated SmeYZ pump function by transcriptionally up-regulating the smeYZQ operon. Nevertheless, in the smeYZ mutant, SmeQ exerted its modulatory role by up-regulating entSCEBB'FA and sbiAB operons, decreasing intracellular iron levels, and causing ΔsmeYZ-mediated pleiotropic defects, except for aminoglycoside susceptibility. SmeQ is the first small protein identified to be involved in efflux pump function in S. maltophilia. It exerts modulatory effect by transcriptionally altering the expression of target genes, which are the smeYZQ operon in the WT KJ, and smeYZQ, entSCEBB'FA and sbiAB operons in smeYZ mutants.

Antibacterial activity of phytochemicals from Bauhinia thonningii significantly increased in the presence of the efflux pump inhibitor, phenylalanine-arginine-β-naphthylamide towards multidrug-resistant phenotypes

Background: View the rapid spread of multidrug-resistant bacteria worldwide; alternatives to antibiotics are necessary for handling various bacterial infections. This study aimed to investigate the antibacterial activity of eight flavonoids isolated from the leaves methanol extract of Bauhinia thonningii against multi-drug resistant (MDR) bacteria expressing efflux pumps. Methods: The antibacterial activity of the phytochemicals alone and in the presence of phenylalanine-arginine-ß-naphthylamide (PAβN) was carried out by the broth micro-dilution method. Spectrophotometric methods were used to evaluate the effects of quercetin-3-O-L-rhamnopyranoside (5) on bacterial growth curve and bacteriolysis, whereas its effect on the bacteria H+-ATPase pumps was monitored by pH measurement. Methods: The antibacterial activity of the phytochemicals alone and in the presence of phenylalanine-arginine-ß-naphthylamide (PAβN) was carried out by the broth micro-dilution method. Spectrophotometric methods were used to evaluate the effects of quercetin-3-O-L-rhamnopyranoside (5) on bacterial growth curve and bacteriolysis, whereas its effect on the bacteria H+-ATPase pumps was monitored by pH measurement. Results: The tested phytochemicals had selective antibacterial activity with minimal inhibitory concentration (MIC) ranging from 8 to 128 μg/mL. Compounds 5, 6, and 8 were active against all the tested bacteria, and compound 8 showed the lowest MIC value (8 µg/mL), especially against E. coli AG102, S. aureus MRSA3, and P. stuartii NEA16. Compound 5 presented good activity (8 < MIC ≤ 32 µg/mL) against 50% of the tested bacteria. Moreover, compounds 5 and 6 have displayed excellent activity (4 < MIC ≤ 32 µg/mL) against P. aeruginosa PA124. In the presence of PAβN, the antibacterial activity of all the tested compounds increased 2 to >128 folds on at least one of the tested bacteria. Quercetin-3-O-L-rhamnopyranoside (5) inhibited bacterial growth in the exponential phase, induced bacteriolysis, and inhibited the H+-ATPase pumps in P. stuartii NEA16. Conclusion: Compounds 5, 6, and 8 isolated from the leaves of Bauhinia thonningii deserve further investigations as potential drugs to fight infections involving MDR bacteria expressing efflux pumps.

Evaluation of Efflux-Mediated Resistance and Biofilm formation in Virulent Pseudomonas aeruginosa Associated with Healthcare Infections

Pseudomonas aeruginosa is a significant pathogen identified with healthcare-associated infections. The present study evaluates the role of biofilm and efflux pump activities in influencing high-level resistance in virulent P. aeruginosa strains in clinical infection. Phenotypic resistance in biotyped Pseudomonas aeruginosa (n = 147) from diagnosed disease conditions was classified based on multiple antibiotic resistance (MAR) indices and analysed with logistic regression for risk factors. Efflux pump activity, biofilm formation, and virulence factors were analysed for optimal association in Pseudomonas infection using receiver operation characteristics (ROC). Age-specificity (OR [CI] = 0.986 [0.946-1.027]), gender (OR [CI] = 1.44 [0.211-9.827]) and infection sources (OR [CI] = 0.860 [0.438-1.688]) were risk variables for multidrug resistance (MDR)-P. aeruginosa infection (p < 0.05). Biofilm formers caused 48.2% and 18.5% otorrhea and wound infections (95% CI = 0.820-1.032; p = 0.001) respectively and more than 30% multidrug resistance (MDR) strains demonstrated high-level efflux pump activity (95% CI = 0.762-1.016; p = 0.001), protease (95% CI = 0.112-0.480; p = 0.003), lipase (95% CI = 0.143-0.523; p = 0.001), and hemolysin (95% CI = 1.109-1.780; p = 0.001). Resistance relatedness of more than 80% and 60% to cell wall biosynthesis inhibitors (ceftazidime, ceffproxil, augumentin, ampicillin) and, DNA translational and transcriptional inhibitors (gentamicin, ciprofloxacin, ofloxacin, nitrofurantoin) were observed (p < 0.05). Strong efflux correlation (r = 0.85, p = 0.034) with MDR strains, with high predictive performances in efflux pump activity (ROC-AUC 0.78), biofilm formation (ROC-AUC 0.520), and virulence hierarchical-clustering. Combine activities of the expressed efflux pump and biofilm formation in MDR-P. aeruginosa pose risk to clinical management and infection control.

Botanicals from nine selected Cameroonian edible plants displayed Anti-pseudomonas effects and potentiated the activities of antibiotics against multidrug-resistant phenotypes over-expressing MexAB-OprM efflux pumps

Background: Despite the recognized efficiency of antibiotic therapy, the annual cases of deaths related to bacterial diseases is still growing in developing countries. In the present study, the in vitro antipseudomonal activity of the methanol extracts of nine food plants from Cameroon against the multidrug-resistant strains and isolates of Pseudomonas aeruginosa overexpressing active efflux pumps was determined. These plants included Persea americana, Syzygium jambos, Mangifera indica, Garcinia kola, Citrus sinensis, Passiflora edulis, Vernonia amygdalina, Aframomum letestuanum, and Artocarpus heterophylus. Methods: The liquid microplate dilution method using the rapid para-Iodonitrotetrazolium chloride (INT) colorimetric method was applied to evaluate the antipseudomonal activities of botanicals, as well as their association with the efflux pump inhibitor and antibiotics. Results: All botanicals displayed an antibacterial activity that varies from one bacterium to another, in the minimal inhibitory concentration (MIC) range of 64 µg/mL to 2048 µg/mL. The extracts from a mixture of leaves and bark of Syzygium jambos and Mangifera indica, the bark of Garcinia kola, and the leaves of Persea americana had the highest spectrum of antipseudomonal activity, with their inhibitory effects being noted in 100% of the 15 tested bacteria. Botanical from the leaves of Garcinia cola, were active against 90% of the strains tested, that from the bark of Persea americana and the leaves of Citrus sinensis were active against 70% and 60% of tested strains and isolates. Botanicals from the leaves and bark of Mangifera indica were very active against the isolates P124 and P57 with a MIC value of 64 µg/mL. At the concentration of MIC/2 and MIC/4, the extract from the leaves of Mangifera indica and Syzygium jambos potentiated the activity of four antibiotics (Penicillin, Ampicillin, Imipenem, Augmentin) on 100% (7/7) of the strains and isolates tested. Persea americana leaf extract also enhanced the activity of penicillin, tetracycline, chloramphenicol, levofloxacin, ampicillin, and augmentin in 85% (6/7) of strains and isolates tested. The activity of all tested antibiotics increased in the presence of botanicals against at least one bacterial strain. The extract of leaves and bark of Persea americana, Psidium guajava, and leaves of Syzygium jambos potentiated the activity of 80% of the antibiotics on the strains and isolates tested. Conclusion: Finally, the methanol extracts from the leaves and bark of Mangifera indica could be used effectively alone or in combination with antibiotics in the treatment of bacterial infection caused by Pseudomonas aeruginosa including antibiotic-resistant phenotypes expressing efflux pumps.

WCK 1152, WCK 1153: Discovery and structure activity relationship for the treatment of resistant pneumococcal and staphylococcal respiratory infections

Novel antibacterial agents needed constantly to counter the ever emergent resistance development to commercially available drugs; one of the effective synthetic antibacterial classes is fluoroquinolone (FQ). This study includes structure activity relationship based design and synthesis of novel fluoroquinolone molecules active against resistant pathogens bearing mutations of DNA gyrase and/or topoisomerase IV which also express efflux pumps. Here, series of compounds were prepared by treating 1-cyclopropyl-6,7-difluoro-8-methoxy-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid as a core with various 4-substituted-3,3-dialkyl piperidines as side chains, through conventional synthetic approaches. Subsequently, antibacterial activities of these fluoroquinolones were examined against Streptococcus pneumoniae, SPN 5844 (Moxi resistant DNA gyrase and topo IV mutant) and SPN 706 (FQ efflux positive). The current manuscript covers >50 examples of fluoroquinolone NCEs, amongst 20 NCEs have shown MIC in the range of (0.4 to >6.25 μg/ml) for SPN 5844 and (0.1–12.5 μg/ml) for SPN 706 strains. During the course of this study; WCK 919, comprising two chiral isomers; WCK 1152 and WCK 1153 were emerged as lead among the different series synthesized. Advance studies suggested either WCK 1152 or WCK 1153 are the worthy candidates for further clinical developments for respiratory infections caused by resistant pneumococci and staphylococci. However, on the basis of in house preclinical work, WCK 1152 had been selected for phase-1 domestic clinical trials.

Serotonin reuptake inhibitors effect on fluconazole activity against resistant Candida glabrata strains

The study aimed to evaluate four selective serotonin reuptake inhibitors (SSRIs) as modifiers of fluconazole activity against resistant strains of Candida glabrata. The effect of SSRIs on fluconazole activity was studied using the checkerboard method against C. glabrata strains (CBS 138, CBS 850821, DSY 562, DSY 565, ATCC 22553 and ATCC 90030); fractional inhibitory concentration index (FIC) was calculated and time-kill curve was used for the most prominent combination for further evaluation. All used SSRIs have antifungal activity against the C. glabrata strains tested. A combination of fluconazole with fluoxetine or fluvoxamine showed indifferent effects (fractional inhibitory concentration index [FICI] in all strains >1 but <4), whereas a paroxetine-fluconazole combination showed an additive effect against DSY565 and CBS138, known to express efflux pumps as well as on ATCC strain (0.5 < FIC < 1) with indifferent effect on other strains used. The most promising combination was that of fluconazole with sertraline (FICI ≤0.5), where a synergistic effect was observed against all resistant and susceptible dose-dependent strains, including those known to express efflux pumps. This synergistic effect was confirmed by time-kill curve assay against all resistant C. glabrata and ATCC strains with a >2-log10 CFU/mL reduction caused by combination compared with a single active agent of fluconazole after 24 hours of incubation. A sertraline-fluconazole combination produced an additive effect on the reference ATCC strain. Our data suggest that blocking active efflux pumps by sertraline may be considered the probable mechanism of synergism with fluconazole. The combination of sertraline with fluconazole could be a promising remedy for treatment of infections caused by resistant C. glabrata.

Phytochemical analysis and antibiotic-modulating activity of Cocos nucifera, Glycine max and Musa sapientum methanol extracts against multidrug resistant Gram-negative bacteria

Background: The rapid emergence of multidrug resistant (MDR) bacteria is occurring worldwide, endangering the efficacy of antibiotics, which have transformed medicine and saved millions of lives. Antibiotic-resistant infections are already widespread in the Sub-Saharan Africa and across the globe. To extend the search for new and more efficient antimicrobial drugs from natural sources, this work has been carried out to study the phytochemical composition and the antibacterial activities of some Cameroonian dietary plants (Cocos nucifera, Glycine max and Musa sapientum) against several MDR Gram-negative strains including Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Klebsiella pneumoniae, Pseudomonas aeruginosa species expressing efflux pumps. Methods: Phytochemical screening of plant extracts was performed using qualitative standard methods and the antimicrobial assays of these extracts alone and in combination with antibiotics were done using serial 96-wells microplate dilution essays. Results: Each plant extract contained at least three mean classes of secondary metabolites. Glycine max, epicarps, leaves and bark of C. nucifera as well as mesocarps of M. sapientum contained each alkaloids, polyphenols, flavonoids, and triterpenes. Moreover, steroids were also found in G. max, steroids and saponins in epicarps and saponins in bark of C. nucifera. Meanwhile epicarps from M. sapientum contained only polyphenols, flavonoids and saponins. Antibacterial assays showed that different parts of C. nucifera were more active than other extracts. Their minimal inhibitory concentrations (MICs) varied from 128 to 2048 µg/mL. The bark part presented the highest antibacterial potential inhibiting the growth of 90% of strains with significant activity (100≤MIC≤512 µg/mL) against 50% of them (three E. coli, four E. aerogenes and three K. pneumoniae). It showed bactericidal effects (MBC/MIC≤4) on 45% of the same bacterial species. It was followed by epicarps and leaves parts which exhibited an inhibitory power against 75% and 60% of bacteria with significant activity on 40% and 20% of them respectively. They also showed bactericidal effects on E. coli ATCC8739 for epicarps extract and E. coli ATCC8739 and P. stuartii NEA16 for leaves extract. Extracts from G. max were less active and those from mesocarps and epicarps of M. sapientum did not showed any activity on all studied bacteria. Bark and epicarps extracts of C. nucifera potentiated the activities of all used antibiotics against at least 70% of bacteria while leaves extract exhibited this effect improving the activities of 67% of antibiotics with improvement activity factors (IAF) ranging from 2 to 256 suggesting that they contain bioactive compounds which could be considered as efflux pumps inhibitors. Extracts from G. max, epicarps and mesocarps of M. sapientum enhanced the inhibitory potential of 56%, 34% and 23% of antibiotics respectively against at least 70% of studied bacteria. These increases of activities also characterize synergistic effects between antibiotics and bioactive compounds of plants. Conclusion: The findings of this work suggest that infections by resistant bacteria can be treated using different parts of C. nucifera as an alternative to commonly used antibiotics.

An Efflux Pumps Inhibitor Significantly Improved the Antibacterial Activity of Botanicals from Plectranthus glandulosus towards MDR Phenotypes.

Bacterial multidrug resistance causes many therapeutic failures, making it more difficult to fight against bacterial diseases. This study aimed to investigate the antibacterial activity of extract, fractions, and phytochemicals from Plectranthus glandulosus (Lamiaceae) against multidrug-resistant (MDR) Gram-negative phenotypes expressing efflux pumps. The crude extract after extraction was subjected to column chromatography, and the structures of the isolated compounds were determined using spectrometric and spectroscopic techniques. Antibacterial assays of samples alone and in the presence of an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide, PAβN) were carried out using the broth microdilution method. The phytochemical study of P. glandulosus plant extract afforded seven major fractions (A–G) which lead to the isolation of seventeen known compounds. The ethanol extract of P. glandulosus was not active at up to 1024 μg/mL, whereas its fractions showed MICs varying from 32 to 512 μg/mL on the studied bacteria. Fraction C of P. glandulosus showed the lowest MIC (32 μg/mL) on E. coli ATCC8739 strain. Fraction D presented the highest activity spectrum by inhibiting the growth of 90% (9/10) of the studied bacteria. The presence of PAβN has improved the activity of extract and all fractions. Overall, the tested phytochemicals showed low activity against the studied bacteria. The overall results obtained in this study show that some fractions from P. glandulosus, mainly fractions C and D, should be investigated more for their possible use to fight against MDR bacteria.

Antibacterial Activities and Phytochemical Screening of Crude Extracts from Kenyan Macaranga Species Towards MDR Phenotypes Expressing Efflux Pumps

Antibacterial Activities and Phytochemical Screening of Crude Extracts from Kenyan Macaranga Species Towards MDR Phenotypes Expressing Efflux Pumps

In vitro antibacterial and antibiotic-potentiation activities of five edible plant extracts and mode of action against several MDR Gram-negative phenotypes

Abstract Background: Bacterial infections involving the multidrug resistant (MDR) strains are among the top leading causes of death throughout the world. Healthcare system across the globe has been suffering from an extra-ordinary burden in terms of looking for the new and more potent antimicrobial compounds. The aim of the present study was to determine the antibacterial activity of some Cameroonian edible plants (Garcinia lucida bark, Phoenix dactylifera pericarps, Theobroma cacao pod, Solanum macrocarpon leaves and Termitomyces titanicus whole plant) and their antibiotics-potentiation effects against some MDR Gram-negative bacteria phenotypes expressing efflux pumps (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Providencia stuartii strains). Methods: The antibacterial activities of plant extract alone and in combination with usual antibiotics were carried out using the micro-dilution method. The effects of the most active plant extract (Garcinia lucida bark) on H+-ATPase-mediated proton pumps and on bacterial growth kinetic were performed using experimental protocols, while qualitative reference methods were used to highligh the major groups of secondary metabolites present in the extracts. Results: Qualitative phytochemical screening of plant extracts indicated that all analysed secondary metabolites were present in Theobroma cacao and Termitomyces titanicus while one (saponins) of them was absent in Garcinia lucida and Solanum macrocarpon. Only three of them (polyphenols, flavonoids and saponins) were detected in Phoenix dactylifera. Antibacterial essays showed that G. lucida was the most active plant as it inhibited the growth of all studied bacteria with strong activity (MIC&lt;100 µg/mL) against E. coli ATCC8739, significant activity (100≤MIC≤512 µg/mL) against 80% of bacteria and moderate activity (512&lt;MIC≤2048 µg/mL) against E. coli AG100A and E. aerogenes (EA289 and CM64). It was followed by T. cacao and S. macrocarpon extracts which exhibited an antibacterial potential against 95% and 80% of bacterial strains, respectively. These three extracts exhibited a bactericidal effect on a few bacteria. Extracts from T. titanicus and P. dactylifera were less active as they moderately (512&lt;MIC≤2048 µg/mL) inhibited the growth of 35% and 10% of bacteria. All extracts selectively potentiated the activities of all antibiotics with improvement activity factors (IAF) ranging from 2 to 256. G. lucida, T. cacao and S. macrocarpon potentiated the activities of 100%, 89% and 67% of antibiotics respectively against more than 70%, suggesting that they contain bioactive compounds which could be considered as efflux pumps inhibitors. Whereas T. titanicus and P. dactylifera improved the activities of almost 40% and 20% of antibiotics, respectively. This increase of activities also characterizes synergistic effects between antibiotics and these bioactive compounds. G. lucida extract at all tested concentrations, strongly inhibited the growth of bacterial strain E. coli ATCC8739 and exhibited an inhibitory effect on this bacterial H+-ATPase-mediated proton pumps increasing the pH of the medium. Conclusion: The overall results indicated that food plants among which G. lucida, T. cacao and S. macrocarpon could have a benefit interest in combatting resistant types of bacteria. Keywords: Food plants; infectious diseases; MDR bacteria; efflux pumps; antibiotics; secondary metabolites.

Antibacterial potential and mechanism of action of botanicals and phytochemicals from Stachytarpheta cayennensis (Verbenaceae) against Gram-negative multidrug-resistant phenotypes expressing efflux pumps

Background: The effectiveness of antibiotics in the fight against bacterial infections motivated their massive use which resulted in the appearance of resistant bacteria. This phenomenon is nowadays in constant evolution and represents a major problem of public health. In this respect, the search for new molecules for fighting against bacterial resistance is essential. […]

Quantitative Imaging of Bacterial Efflux Pumps through Single-Molecule Localization Microscopy

One of the most successful strategies that bacteria evolved to resist antimicrobial treatments is that of expressing efflux pumps (EPs), a class of transporter proteins capable of extruding a wide range of molecules from their hosts, including harmful metabolic products and xenobiotics. Although these proteins are extremely relevant in clinical scenarios, much is still unclear about the regulation dynamics of EPs and their spatial distribution within and between bacterial cells. Due to traditional gene expression assays being performed on bulk samples such as plated colonies or broth cultures, the experimental data we have is a global average obtained from billions of individual bacteria after long incubation times. We lack the ability to (i) detect gene expression changes occurring on short timescales, and to (ii) do so with subcellular spatial resolution. Subtle changes in the expression pattern of a given protein would be apparent only if the measurement had a very high precision - ideally, on the order of the single protein. Furthermore, in order to acquire any valid information about spatial organization in complex, three-dimensional communities such as biofilms, we need to be able to obtain our data without perturbing the overall structure of the samples. Here, we present a quantitative PhotoActivated Localization Microscopy (qPALM) approach to investigate the expression dynamics and the distribution of the AcrB efflux pump component in E. coli. By labelling the endogenous protein with a photoactivatable marker (PAmCherry) via CRISPR/Cas9-assisted genome editing, we are able to localize and count individual instances of AcrB in bacterial cells through single-molecule localization microscopy while maintaining physiological copy numbers. We validated our method by measuring AcrB expression after treatment with known inducers and inhibitors of the gene, and we are now applying it to investigate AcrB distribution in biofilms.

Evaluation of four calcium channel blockers as fluconazole resistance inhibitors in Candida glabrata.

The aim of this study was to evaluate the ability of four calcium channel blockers (CCBs), namely verapamil, diltiazem, nicardipine (NIC) and nifedipine (NIF), to enhance the susceptibility of Candida glabrata strains to fluconazole (FLC). Synergistic antifungal effects of the CCBs with FLC were examined by the chequerboard method, and fractional inhibitory concentration indices (FICIs) were determined. The time-kill curve method was used for the most promising combination to further evaluate the synergetic effects. NIC showed an additive effect with FLC against FLC-resistant and FLC-susceptible-dose-dependent strains (DSY 565 and CBS 138) known to express efflux pumps, but not against FLC-susceptible strains. NIF exhibited an additive effect with FLC both by the chequerboard method (0.5<FICI<1) and time-kill curves (<2 log10 CFU/mL decrease in viable count). In addition, NIF had its own antifungal effect consistently against most of the strains used in this study, with minimum inhibitory concentrations (MICs) of 8μg/mL. NIC showed an additive effect with FLC against FLC-resistant C. glabrata strains, most probably via efflux pump inhibition as demonstrated selectively in FLC-resistant strains with known efflux pumps. NIF displayed a promising antifungal effect alone as well as an additive effect with FLC against most of the strains.

Morphological changes and differentially expressed efflux pump genes in Mycobacterium tuberculosis exposed to a rifampicin and verapamil combination

The aim of the present study was to (i) evaluate the in vitro action of rifampicin (RIF), ethambutol or isoniazid with efflux pumps inhibitors (EPIs) in Mycobacterium tuberculosis (Mtb) H37Rv and (ii) evaluate the morphological and efflux pumps (EPs) transcriptional changes by the action of rifampicin + verapamil combination (RIF + VP). The minimal inhibitory concentration and synergic effect of drug combinations were determined by Resazurin Microtiter Plate Assay and Resazurin Drugs Combination Microtiter Assay, respectively. VP showed greater capacity of ethidium bromide accumulation and RIF + VP had the lower fractional inhibitory concentration index. The RIF + VP exerted a similar reduction of viable cell counts to RIF by time-kill curve, but decreases in the expression of EPs genes were observed by Real time PCR at 72 h of RIF + VP exposure. Accumulative morphological changes (wrinkled and rounding) caused by each drug were observed by scanning electron microscopy after RIF + VP exposure. The downexpression of EPs related genes exposed to RIF + VP, suggest an effective inhibitory activity of VP in Mtb H37Rv. The role of EPs and the use of EPIs open up a powerful approach and the RIF + VP combination should be studied in Mtb more thoroughly.

Inhibitors of the Candida albicans Major Facilitator Superfamily Transporter Mdr1p Responsible for Fluconazole Resistance.

ObjectiveTo identify a novel class of inhibitors of fungal transporters involved in drug resistance.MethodsA series of structurally-related low molecular mass compounds was synthesized using combinatorial chemistry of a cyclobutene-dione (squarile) core. These compounds were screened for their inhibition of plasma membrane Major Facilitator Superfamily (MFS) and ATP-binding cassette (ABC) transporters responsible for efflux pump-mediated drug resistance in the fungal pathogen Candida albicans. Strains of Saccharomyces cerevisiae that specifically overexpress the MFS pump CaMdr1p or the ABC transporter CaCdr1p were used in primary screens and counterscreens, respectively, and to detect inhibition of glucose-dependent Nile Red efflux. Efflux pump inhibition, activity as pump substrates and antifungal activity against yeast and clinical isolates expressing efflux pumps were determined using agarose diffusion susceptibility assays and checkerboard liquid chemosensitization assays with fluconazole.ResultsThe screen identified five structurally-related compounds which inhibited CaMdr1p. Two compounds, A and B, specifically chemosensitized AD/CaMDR1 to FLC in a pH-dependent fashion and acted synergistically with FLC in checkerboard liquid MIC assays but compound B had limited solubility. Compound A chemosensitized to FLC the azole-resistant C. albicans strain FR2, which over-expresses CaMdr1p, inhibited Nile Red efflux mediated by CaMdr1p but not CaCdr1p and was not toxic to cultured human cells. A minor growth-inhibitory effect of B on AD/CaMDR1, but not on AD/CaCDR1 and AD/CaCDR2, indicated that compound B may be a substrate of these transporters. The related compound F was found to have antifungal activity against the three pump over-expressing strains used in the study.ConclusionsCompound A is a ‘first in class’ small molecule inhibitor of MFS efflux pump CaMdr1p.

Synthetic biology applications in industrial microbiology

Synthetic biology applications in industrial microbiology

Antibiotic-potentiation activities of four Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps.

BackgroundThe continuous spread of multidrug-resistant (MDR) bacteria, partially due to efflux pumps drastically reduced the efficacy of the antibiotic armory, increasing the frequency of therapeutic failure. The search for new compounds to potentiate the efficacy of commonly used antibiotics is therefore important. The present study was designed to evaluate the ability of the methanol extracts of four Cameroonian dietary plants (Capsicum frutescens L. var. facilulatum, Brassica oleacera L. var. italica, Brassica oleacera L. var. butyris and Basilicum polystachyon (L.) Moench.) to improve the activity of commonly used antibiotics against MDR Gram-negative bacteria expressing active efflux pumps.MethodsThe qualitative phytochemical screening of the plant extracts was performed using standard methods whilst the antibacterial activity was performed by broth micro-dilution method.ResultsAll the studied plant extracts revealed the presence of alkaloids, phenols, flavonoids, triterpenes and sterols. The minimal inhibitory concentrations (MIC) of the studied extracts ranged from 256-1024 μg/mL. Capsicum frutescens var. facilulatum extract displayed the largest spectrum of activity (73%) against the tested bacterial strains whilst the lower MIC value (256 μg/mL) was recorded with Basilicum polystachyon against E. aerogenes ATCC 13048 and P. stuartii ATCC 29916. In the presence of PAβN, the spectrum of activity of Brassica oleacera var. italica extract against bacteria strains increased (75%). The extracts from Brassica oleacera var. butyris, Brassica oleacera var. italica, Capsicum frutescens var. facilulatum and Basilicum polystachyon showed synergistic effects (FIC ≤ 0.5) against the studied bacteria, with an average of 75.3% of the tested antibiotics.ConclusionThese results provide promising information for the potential use of the tested plants alone or in combination with some commonly used antibiotics in the fight against MDR Gram-negative bacteria.

Investigation of the expression levels of efflux pumps in fluconazole-resistant Candida albicans isolates

Widespread and repeated use of fluconazole in the prophylaxis and therapy resulted in resistance among Candida strains. Investigation of the expression of efflux pump encoding genes was aimed in fluconazole-resistant and -susceptible C.albicans isolates in order to determine the role of this mechanism in fluconazole resistance. Five fluconazole-resistant, six -susceptible and four trailing effect showing susceptible C.albicans isolates were included in the study. The MIC values of fluconazole and other antifungal agents were determined by the microdilution method. The fluconazole MIC values of the fluconazole-resistant strains were also studied by E-test performed on yeast extract peptone dextrose agar with and without cyclosporin A. The expression levels of CDR1, CDR2 and MDR1 transcripts were determined by real-time PCR method. The expression of these genes was normalized with their ACT1 levels and compared with the fluconazole-susceptible C.albicans ATCC 14053 strain. It was detected that all strains were susceptible to amphotericin B and all except one strain were also susceptible to clotrimazole. Three out of five fluconazole-resistant strains and three out of four trailing effect showing susceptible strains were resistant to 5-flucytosine, and all except one susceptible strains were found as intermediate to 5-flucytosine. All except one fluconazole-resistant strains were determined as resistant to itraconazole and ketoconazole, and had miconazole MIC values of ≥ 64 µg/ml. All fluconazole-susceptible isolates were detected to be susceptible to ketoconazole and dose dependent susceptible to itraconazole. Fluconazole-resistant and -susceptible strains were determined as susceptible to voriconazole. Out of five fluconazole-resistant isolates, two strains overexpressed high levels and three strains overexpressed mild levels of CDR1/2; one strain overexpressed high levels and three strains overexpressed low levels of MDR1 in comparison to C.albicans ATCC 14053 control strain. It was observed that CDR1, CDR2 and MDR1 gene expression levels were mild in strains showing trailing effect except one which highly expressed MDR1, while susceptible isolates except three expressed efflux pump genes at low levels. It was determined that the expression levels of CDR1 and CDR2 genes for the same strain were in parallel for all isolates. It can be concluded that overexpression of efflux pump genes is an important mechanism of resistance in fluconazole-resistant C.albicans isolates.